Vascular tissue engineering

ABSTRACT

The invention relates to tubularized tissue, which in one embodiment is a vascular tissue made by seeding cells on the exterior surface of a scaffold and incubating of the cells so as to form a tubular tissue with the scaffold inside. In another embodiment the invention relates to a hybrid tissue. In another embodiment, the invention relates to methods of using the same for example, for enhancing blood vessel formation in a patient, for promoting angiogenesis and vasculogenesis, for replacing damaged blood vessel and for determining cellular or tissue function of an agent.

[0001] This application is a Continuation-in Part Application ofPCT/IL02/00336, filed Apr. 30, 2002 which claims priority fromprovisional Application No. 60/287,003, filed Apr. 30, 2001, which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Blood vessels are the means by which oxygen and nutrients aresupplied to living tissues and waste products removed from livingtissue.

[0003] The need for re-vascularization is emphasized in diabeticpatients. 143 million people suffer from Diabetes, worldwide, and theirnumber is estimated to be more than doubled by the year 2025. In theU.S. alone, 200,000-400,000 diabetic patients develop foot ulcers due topoor blood flow to the extremities. These ulcers might turn into footgangrene. From 1993 to 1995, about 67,000 amputations were performedeach year among diabetic patients.

[0004] Vascular development occurs in two stages: a) an early stage ofvasculogenesis, by which the primary capillary network is formed frommesoderm-derived precursors, hemangioblasts, through a process ofdifferentiation and proliferation in situ within a previously avascularorgan or tissue and b) a coalescence of these cells to form a primitivetubular network. The later stage has been termed angiogenesis, whichrefers to the formation of new capillary vessels from pre-existingmicro-vessels by remodeling and maturation of the primary plexus. Abalance between pro and anti-angiogenic molecules regulates thisprocess. While vasculogenesis occurs primarily during earlyembryogenesis, angiogenesis in the adult occurs as a stage of everyinflammatory process (1,2).

[0005] Angiogenesis is the process by which new blood vessels are formed(Folkman and Shing, J. Biol. Chem. 267 (16), 10931-10934 (1992). It isessential in reproduction, development and wound repair. However,inappropriate angiogenesis can have severe consequences. For example, itis only after many solid tumors are vascularized as a result ofangiogenesis that the tumors begin to grow rapidly and metastasize. Theangiogenesis process is believed to begin with the degradation of thebasement membrane by proteases secreted from endothelial cells (EC)activated by mitogens such as vascular endothelial growth factor (VEGF)and basic fibroblast growth factor (bFGF). The cells migrate andproliferate, leading to the formation of solid endothelial cell sproutsinto the stromal space, then, vascular loops are formed and capillarytubes develop with formation of tight junctions and deposition of newbasement membrane.

[0006] There is a wide recognition that there is a great clinical needfor a readily available, small or medium diameter vascular graft. Thesegrafts are especially required in cardiology for atheroscelerotic bloodvessels replacement and ischemic heart treatment. Approximately 12.6million people alive today have a history of heart attack, angina, orboth. Arterial replacement is a common treatment for this disease.Autogenous vessels, particularly internal mammary arteries and saphenousveins, remain the “gold standard” for coronary grafting. However, 30% ofthe patients in need of arterial bypass do not have veins suitable forgrafting due to diseased veins. In addition, significant morbidity,surgical costs and restenosis have been associated with the harvest ofautologous vessels.

[0007] In addition, in the presence of an injury or a defect in otherorgans in the body, surgical approaches to correcting defects in thebody, in general, involve the implantation of structures made ofbiocompatible, inert materials, that attempt to replace or substitutefor the defective function. Non-biodegradable materials will result inpermanent structures that remain in the body as a foreign object.Implants that are made of resorbable materials are suggested for use astemporary replacements where the object is to allow the healing processto replace the resorbed material. However, these approaches have metwith limited success for the long-term correction of structures in thebody.

[0008] Therefore, there is a need for developing tubular tissues, inorder to transplant or replace narrow and/or thick vessels such as ablood vessel or tracts such as gastrointestinal tract and genitourinarytract, whereby the scaffold will be degradated after transplantationinto the body.

SUMMARY OF THE INVENTION

[0009] The invention relates to cells which are grown on the exteriorsurface of a scaffold in a way permitting tubularized tissue with adegradable scaffold contained by methods of using the same fortransplanting or replacing damaged tracts or vessels in the body andmethods of using the same for diagnostic or screening purposes.

[0010] In one embodiment, the invention provides a tubularized tissuemade according to the steps of: seeding a cell on the exterior surfaceof scaffold so as to obtain a scaffold encircled by cells; incubatingsaid scaffold encircled by said cells so as to form a tubularizedtissue; thereby obtaining a tubularized tissue.

[0011] In another embodiment, the invention provides a method ofmaturing cells into a tubularized tissue comprising the following steps:seeding a cell on the exterior surface of scaffold so as to obtain ascaffold encircled by cells; incubating said scaffold encircled by saidcells so as to form a tubularized tissue; thereby obtaining atubularized tissue.

[0012] In another embodiment, the invention provides a method ofenhancing blood vessel formation in a patient in need thereof,comprising the following steps: selecting the patient in need thereof;isolating a cell from the patient; seeding a cell on the exteriorsurface of scaffold so as to obtain a scaffold encircled by cells;incubating said scaffold encircled by said cells so as to form atubularized tissue; and re-administering said tubularized tissue to thepatient in need thereof, thereby enhancing blood vessel formation in apatient in need thereof.

[0013] In another embodiment, the invention provides a hybrid tissuemade according to the steps of: seeding a cell that will form a firsttissue type on the exterior surface of a branched scaffold so as toobtain a scaffold encircled by cells with spaces in between; adding intosaid spaces in between, a cell that will form a second tissue type so asto form a hybrid tissue; and incubating so as to obtain a hybrid tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1: b-End-2 Endothelial Cell Line Expressing GFP, In VitroCharacterization and In Vivo Detection. a) b-End-2 cells in 3D ECMculture, show endothelial cells (ECs) phenotype, forming tubularstructures. (b), Retro-GFP transduction (MOI: 5) of b-End-2 ECs, yields94% transduction efficiency detected by FACS analysis. (c) b-End-2 ECsinduce spontaneous formation of blood containing cavities (arrows) wheninjected s.c. in CD1 nude mouse ear, (d) Fluorescence microscopy ofcultured transduced b-End-2 ECs, reveals GFP expression. (e) GFPpositive cells (b-End-2) detected in vivo (s.c. transplanted, 70 days)by immnunohistochemistry. (e-bottom) negative control.

[0015]FIG. 2: Filament-Like Polymeric Scaffold Product (polyesteranhydrate) Based on Lactic, Glycolic and Ricinoleic Acids

[0016]FIG. 3: b-End-2 Endothelial Cells Cultured on Filament-likePolymeric Scaffold in Rotary Bioreactor (day 14). (a) Microphotograph;and Histology (b) H&E of endothelialized filament like polymericscaffold indicate formation of primitive micro-capillary networkstructure in 14 days.

[0017]FIG. 4: b-End-2 Endothelial Cells Cultured on Filament-likePolymeric Scaffold Form a Primitive Micro-capillary Network Ex Vivo, andIn Vivo (day 28) (a) Microphotograph of S.C. transplant, 2 weeks aftertransplantation; (b) Histology H&E, of transplanted: (c) b-End-2 ECscultured on filament-like polymeric scaffold; b-End-2 ECs culturedwithout polymeric scaffold; (d) naked filament-like polymeric scaffold.P: filament-like Polymeric scaffold; Arrows: b-End-2 ECs.

[0018]FIG. 5: b-End-2 ECs Cultured 7 days in Rotary Bioreactor onFilament-like Polymeric Scaffold, Transplanted S.C. into CD1 Nude Mousefor 42 days. (a) Microphotograph of s.c.transplaant d.42, showing bloodvessels (BV) in the implant; (b) Frozen sections GFP Fluorescentmicroscopy and (c) H&E Histochemistry, showing co-localized b-end-2 ECsadjacent to filament-like polymeric scaffold (arrows). (d) Histology H&Eon paraffin sections, showing b-End-2 ECs (arrows) lining thefilament-like polymeric scaffold (P).

[0019]FIG. 6: b-End-2 ECs Cultured 7 days in Rotary Bioreactor onFilament-like Polymeric Scaffold, Transplanted S.C. into CD1 Nude Mousefor 56 days. (a) Microphotograph of s.c.transplant d.56, showing bloodvessels (BV) in the implant; (b) Histology H&E (c,d) and ReticulumStaining. Note: monolayer of b-End-2 ECs (arrows) lining thefilament-like polymeric scaffold (P), Erythrocytes (asterisks) areevident in the lumen formed due to degradation of polymeric scaffold andbasement membrane (BM) surrounding endothelial cells (d). (c) Reticulumstaining of the host blood vessel basement membrane as a positivecontrol, showing the appearance of basement membrane in mature bloodvessel.

[0020]FIG. 7: MRI Analysis of Implant Vessel Functionality (VF):b-End-2Endothelial Cells Cultured 60 days in Rotary Bioreactor, TransplantedS.C. into CD1 Nude Mouse for 5 days. Representative coronal gradientecho image of the implant. Increased implant vascularity is reflected byreduction of the mean signal intensity (dark area inside the implant).Note: vascularization (arrows), and the increased vessel functionality(VF) in the implant.

[0021]FIG. 8: b-End-2 endothelial cells cultured 7 days in bioreactor,transplanted S.C. into CD1 nude mouse for 28 days (H&E staining and antiCD31 [PECAM]/paraffin sections).

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0022] The invention relates to cells which are grown on the exteriorsurface of a scaffold permitting a tubularized tissue with a scaffoldcontained by, methods of using the same for transplanting or replacingdamaged tracts or vessels in the body and methods of using the same fordiagnostic or screening purposes. In another embodiment, the inventioncan be used to generate a hybrid tissue grafts composed of at least twotissue types co-arranged in a functional architecture.

[0023] The invention relates to cells, which are grown so as to from atubularized tissue analogous to tissue counterparts in vivo.

[0024] The term “tubularized tissue” refers hereinabove to a tissuewhich is in a form of a tube such as vessels, for example without beinglimited, blood vessels, or tracts such as for example genitourinarytract or gastrointestinal tract, tissues for hernia repair, tendons andligaments.

[0025] The different biological structures described below have severalfeatures in common. They are all tubular structures primarily composedof layers of stromal tissue with an interior lining of epithelium(gastrointestinal and genitourinary) or endothelium (blood vessels).Their connective tissues also contain layers of smooth muscle withvarying degrees of elastic fibers, both of which are especiallyprominent in arterial blood vessels. By including and sustaining thesecomponents in the tubular tissue according to the invention, the tissuesthey compose can attain the special structural and functional propertiesthey require for proper physiological functioning in vivo. They can thenserve as replacements for damaged or diseased tubular tissues in aliving body.

[0026] Arteries—Arteries are tubes lined with a thin layer ofendothelial cells and generally composed of three layers of connectivetissue: the intima (which is not present in many muscular arteries,particularly smaller ones), media, and adventitia, in order from insideto outside.

[0027] The main cellular component of the inner two layers is anundifferentiated smooth muscle cell, which produces the extracellularprotein elastin. The internal elastic lamina, which lies just interiorto the media, is a homogenous layer of elastin. The abundance of elastinin their walls gives arteries the ability to stretch with everycontraction of the heart. The intima and media also contain somefibroblasts, monocytes, and macrophages, as well as some collagen. Theadventitia is composed of more ordinary connective tissue with bothelastic and collagenic fibers. Collagen in this layer is important inpreventing over-stretching. While all the layers of the arterial wallare connective tissue, there is a compositional and functionaldifference between the adventitia and the inner two coats, the intimaand the media. Consequently, it may be advantageous in accordance withthe invention to grow these different layers in separate meshes. Whetherthe intima and media are grown in separate meshes, or combined in one,depends on how distinct these layers are in the particular artery intowhich the tubularized tissue is to be implanted.

[0028] For example, according to the invention fibroblasts can beisolated from the adventitia of a patient's artery and used to inoculatea three-dimensional matrix, and grown to sub-confluence. Cells can beisolated from tissue rich in elastin-producing undifferentiated smoothmuscle cells, also containing some fibroblasts, from the intima andmedia of the same artery. Endothelial cells can be isolated from thesame patient.

[0029] Veins—The layers of the connective tissue comprising the walls ofveins are less well delineated than those of arteries, and contain muchmore collagen and less elastin. Consequently, a single three-dimensionalculture can be grown, for example, from a single inoculum of cells.These cells consisting mostly of fibroblasts with some smooth musclecells, can be isolated from the walls of a vein of the patient. When theappropriate degree of confluence is reached, endothelial cells, isolatedfrom the same patient, for example, can be seeded on top of the stromallayer and grown to confluence.

[0030] The gastrointestinal tract comprises several different organs,but all have the same general histological scheme.

[0031] Mucous Membrane: The mucous membrane is the most interior layerof the gastrointestinal tract, and is composed of three sub-layers. Theabsorptive surfaces particularly are highly folded to increase thesurface area. The lumen is lined with a thin layer of epithelium, whichis surrounded by the lamina propria, a connective tissue which containsfibroblasts, some smooth muscle, capillaries, as well as collagenic,reticular, and some elastic fibers. Lymphocytes are also found here toprotect against invasion, especially at absorptive surfaces where theepithelium is thin. The third sub-layer, the muscularis mucosa, consistsof two thin layers of smooth muscle with varying amounts of elasticfibers. The smooth muscle fibers of the inner layer are arrangedcircularly, and the outer layer is arranged longitudinally.

[0032] Submucosa: This layer consists of loose connective tissueincluding elastic fibers as well as larger blood vessels and nervefibers.

[0033] Muscularis Externa: This layer consists of two thick layers ofsmooth muscle, providing the motion, which advances material alongthrough the gastrointestinal tract. The muscle fibers of the inner layerare arranged circularly, while in the outer layer they are longitudinal.An exception is the upper third of the esophagus, which containsstriated muscle allowing for the voluntary contractions associated withswallowing.

[0034] Serosa (or Adventitia): This outermost layer consists of looseconnective tissue, covered by squamous mesothelium where the tract issuspended freely.

[0035] Ureter—Like the gastrointestinal tract, the ureter also has amucous membrane as its inner layer. Despite not having an absorptivesurface, the interior surface of the ureter is highly folded to form astellate conformation in cross-section. The epithelial lining, however,is four to five cells thick. The lamina propria, which lies beneath theepithelium, contains abundant collagen, some elastin, and occasionallymph nodules.

[0036] Surrounding the mucous membrane is a muscular coat, whose innerlayer contains longitudinally arranged smooth muscle fibers, while thoseof the outer layer are circularly arranged. The outermost layer, theadventitia, consists of fibroelastic connective tissue.

[0037] Urethra—The urethra consists simply of a lamina propria, which islined with epithelium and surrounded by two layers of smooth musclefibers. In the inner layer, the fibers are arranged longitudinally,while in the outer layer they are circular. The connective tissue of thelamina propria is rich in elastic fibers and contains many venules.Since the urethra has only one stromal layer, a monolayer culture maysuffice for its construction in vitro in accordance with the invention.

[0038] In one embodiment, the invention provides a tubularized tissueprepared according to the steps of: obtaining a cell; obtaining ascaffold; seeding said cell exterior to said scaffold so as to obtain ascaffold encircled by cells; growing said scaffold encircled by saidcells so as to form a tubularized tissue with said scaffold containedby, so as to support said tubularized tissue; thereby obtaining atubularized tissue. Upon administration of the tubularized tissue withthe scaffold contained by, the scaffold is slowly degraded so as toenable the formation of the lumen inside the tubular tissue.

[0039] The cells of the present invention are supported by a scaffold,which in one embodiment is in a form of a tube.

[0040] In another embodiment, the scaffold can be in differentdiameters, according to the use. The following are examples to thevarious diameters of different elements in the vascular systems: Elasticartery >1 cm Muscular artery (large) 2-10 mm Muscular artery (small)0.1-2 mm Arteriole 10-100 um Capillary 4-10 um Post capillary venule10-50 um Muscular venule 50-100 um Small vein 0.1-1 mm Medium vein 1-10mm Large vein >1 cm

[0041] The dimensions of a red blood cell range from 6 to 10um(diameter)×2.6 um. The diameters can vary from 5 um to 300 um, for theformation of a narrow blood vessel or a thick blood vessel,respectively, wherein the artery diameter is 10 um->1 cm, the diameterof a vein is 10 um->1 cm and the capillary diameter is in the range of4-10 um. In another embodiment the invention prodes also a method forthe formation or repair of a lymphatic vessel. In another embodiment,the invention provides a method for the formation of small vesselswithout blocking the lumen.

[0042] In another embodiment, the invention provides a method for theformation of capillary bed by obtaining a cell; obtaining a scaffold;seeding said cell exterior to said scaffold so as to obtain a scaffoldencircled by cells; growing said scaffold encircled by said cells so asto form a capillary bed with said scaffold contained by, so as tosupport said capillary bed; thereby obtaining a capillary bed. Uponadministration of the capillary bed tissue with the scaffold containedby, the scaffold is slowly degraded so as to enable the formation of thelumen inside the capillary bed.

[0043] In another embodiment, the invention provides a method for theformation or repair of a bile duct and in another embodiment theinvention provides a method for the formation or repair of a pancreaticduct (main and accessory).

[0044] In another embodiment, the tubularized tissue of the inventionmay be the large intestine. In this embodiment, the diameter of thescaffold should be between 4-8 cm. In another embodiment, thetubularized tissue may be the small intestine. The diameter of thescaffold for this embodiment is 1.5-3.5 cm.

[0045] In one embodiment, the tubularized tissue is a monolayer tubulartissue. For the formation of a capillary for example, a monolayertubular tissue is required, made of endotelial cells.

[0046] In another embodiment the tubularized tissue is a multi-layertubularized tissue comprising more than one type of cells. The term“multi-layer” refers hereinabove to more than one layer and is use. Forobtaining such a multi-layer tubularized tissue it is required either toseed more than one type of cells on the scaffold or in anotherembodiment to grow the first layer till confluence and than to addanother type of cell on top of the first layer. For a capillaryformation, for example, a first cell layer of endotelial cells isrequired, whereas the second layer comprises pericytes. In the case of aGI tract the inner layer will be a epithelial cell and the outer layerwill be smooth muscle cells arranged in to layers of circular andlongitudinal orientations. The same for urinary, bile and lymph vessels.In accordance with the invention, stromal cells, such as fibroblasts,are inoculated and grown on a exterior to a scaffold, wherein thescaffold is contained by. The scaffold may be configured into the shapeof the connective structure desired. Stromal cells may also includeother cells found in loose connective tissue such as smooth musclecells, endothelial cells, macrophages/monocytes, adipocytes, pericytes,reticular cells found in bone marrow stroma, chondrocytes, etc.

[0047] In another embodiment, the tubularized tissue is a vascularizedtissue. The invention enables the formation of blood capillary, arteryor vein either as a single vessel, or several vessels or a capillarynetwork. For the capillary network, the scaffold has to be structuredwith a number of branches so as to enable a network, which is thantransplanted into the body. For the preparation of a capillary bloodvessel, a monolayer comprises endotelial cells is required. For thepreparation of a capillary, a first layer of endotelial cells isrequired with at least one layer of pericytes on top of the endotelialcells. In another embodiment, it is possible to transplant a monolayertubularized tissue and the pericytes from the body will than attached tosaid monolayer tubularized tissue upon its introduction to the body,whereas the first layer is served as an adherent Layer.

[0048] In another embodiment, the newly formed tissue can be a hybridtissue. The term “hybrid tissue” is referred herein under to a tissue,which is composed of cells which are differentiated to form at least twodifferent tissues. An example for an hybrid tissue is, without beinglimited, vacularized-bone, vacularized-liver and the like. The hybridtissue can grow in one embodiment on top of a scaffold. According tothis embodiment at least one layer first cell type, for example withoutlimitation, a cell that will be differentiated into a vascular tissue,will grow on the top of the scaffold and at least another cell type willgrow on top of the first cell type. The other cell type can be withoutlimitation a bone or a liver. In another embodiment, the scaffold is abranched scaffold. The first cell type, grown on the branched scaffoldwill create a network of cells, with spaces in between, wherein theother cell type will grow said spaces.

[0049] For obtaining the three dimensional tubularized tissue the cellsare seeded on the exterior surface of the scaffold and are incubated ina bioreactor. Other methods for obtaining three-dimensional tissue arestatic tissue culture dish, tissue culture dish placed on a shaker, atube placed on a shaker and a spinner flask.

[0050] The cells of the invention can be any cell derived from a bloodvessel (artery, vein or capillary) or a tract (the genitourinary tractor the gastrointestinal tract) the body.

[0051] In another embodiment the cell used in the invention is amesenchymal stem cell, mesodermal progenitor cell, endothelial precursorcell or neonatal dermal micro vascular endothelial cells (foreskin). Inanother embodiment, at least for the first layer, or for a tissuecomposed of a monolayer of cells, the cell is capable of differentiatinginto an endothelial cell.

[0052] For the GI tract the inner cells are not endothelial cells, thecells are mainly epithelial cells.

[0053] In another embodiment, the cell is isolated from peripheralblood. In one embodiment the cell is obtained from the subject in needand than is re-administered to the subject after being grown into atubular tissue. In another embodiment, the invention provided apossibility of a universal donor, i.e. a bank of tubular tissues atvarious sized and diameters for a universal use. This can be done forexample with cells, which are known as non-immunogenic such as bonemarrow/peripheral blood MSC, and the neonatal dermal micro vascularendothelial cells (foreskin) cell.

[0054] To obtain the EC progenitors from peripheral blood about 5 ml toabout 500 ml of blood is taken from the patient. In another embodimentabout 50 ml to about 200 ml of blood is taken.

[0055] EC progenitors can be expanded in vivo by administration ofrecruitment growth factors, e.g., GM-CSF and IL-3, to the patient priorto removing the progenitor cells.

[0056] In another embodiment, the cell of the invention can beengineered in one embodiment to express a therapeutic agent and/or inanother embodiment to express macromolecules necessary for cell growth,morphogenesis, differentiation, and tissue building can also be added tothe biopolymer molecules or to the biopolymer fibrils to further promotecell in growth and tissue development and organization of the cells.

[0057] The term “macromolecules necessary for cell growth,morphogenesis, differentiation, and tissue building” refers to thosemolecules, e.g., macromolecules such as proteins, which participate inthe development of tissue. Such molecules contain biological,physiological, and structural information for development orregeneration of the tissue structure and function. Examples of thesemacromolecules include, but are not limited to, growth factors,extracellular matrix proteins, proteoglycans, glycosaminoglycans andpolysaccharides. Alternatively, the scaffold can include extracellularmatrix macromolecules in particulate form or extracellular matrixmolecules deposited by cells or viable cells.

[0058] The term “growth factors” is art recognized and is intended toinclude, but is not limited to, one or more of platelet derived growthfactors (PDGF), e.g., PDGF AA, PDGF BB; insulin-like growth factors(IGF), e.g., IGF-I, IGF-II; fibroblast growth factors (FGF), e.g.,acidic FGF, basic FGF, beta.-endothelial cell growth factor, FGF 4, FGF5, FGF 6, FGF 7, FGF 8, and FGF 9; transforming growth factors (TGF),e.g., TGF-P1, TGF-beta.1.2, TGF-beta 2, TGF-beta 3, TGF-beta 5; bonemorphogenic proteins (BMP), e.g., BMP 1, BMP 2, BMP 3, BMP 4; vascularendothelial growth factors (VEGF), e.g., VEGF, placenta growth factor;epidermal growth factors (EGF), e.g., EGF, amphiregulin, betacellulin,heparin binding EGF; interleukins, e.g., IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14; colonystimulating factors (CSF), e.g., CSF-G, CSF-GM, CSF-M; nerve growthfactor (NGF); stem cell factor; hepatocyte growth factor, and ciliaryneurotrophic factor. The term encompasses presently unknown growthfactors that may be discovered in the future, since theircharacterization as a growth factor will be readily determinable bypersons skilled in the art.

[0059] The theraputic agent can be selected for example without beinglimited anti-infective, a hormone, an analgesic, an anti-inflammatoryagent, a chemotherapeutic agent, an anti-rejection agent, aprostaglandin, RGD peptide and combinations thereof.

[0060] For anti-neoplastic therapies, for example, the cells can betransfected with or coupled to cytotoxic agents, cytokines orco-stimulatory molecules to stimulate an immune reaction, otheranti-tumor drugs or angiogenesis inhibitors. For treatment of regionalischemia, angiogenesis could be amplified by prior transfection of cellsto achieve constitutive expression of angiogenic cytokines and/orselected matrix proteins. In addition, the cells may be labeled, e.g.,radio-labeled, administered to a patient and used in the detection ofischemic tissue or vascular injury.

[0061] The cells can also be used to deliver genes to enhance theability of the immune system to fight a particular disease or tumor. Forexample, the cells can be used to deliver one or more cytokines (e.g.,IL-2) to boost the immune system and/or one or more antigens.

[0062] These cells may also be used to selectively administer drugs,such as an antiangiogenesis compound such as O-chloroacetyl carbamoylfumagillol (TNP-470). Preferably the drug would be incorporated into thecell in a vehicle such as a liposome, a timed released capsule, etc. Thecells would then selectively home in on a site of active angiogenesissuch as a rapidly growing tumor where the compound would be released. Bythis method, one can reduce undesired side effects at other locations.

[0063] In another embodiment, the invention provides a method ofrecovering and remodeling or supporting damaged vessels. The tubularizedtissue formed by the method of the invention can be used as a supportfor the damaged tissue, wherein the cells may comprise in one embodimentnucleic acid sequences that encodes to a protein which is active indegradation of without being limited a plaque, LDL.

[0064] The cells used for obtaining the tubularized tissue of theinvention may also be modified ex vivo. For example without beinglimited the cells can be engineered inhibit or enhance angiogenesis.This can be accomplished, for example, by introducing DNA encodingangiogenesis inhibiting agents to the cells, using for example the genetransfer techniques mentioned herein. Angiogenesis inhibiting agentsinclude, for example, proteins such as thrombospondin (Dameron et al.,Science 265:1582-1584 (1994)), angiostatin (O'Reilly et al., Cell79:315-328 (1994), IFN-alpha (Folkman, J. Nature Med. 1:27-31 (1995)),transforming growth factor beta, tumor necrosis factor alpha, humanplatelet factor 4 (PF4); substances which suppress cell migration, suchas proteinase inhibitors which inhibit proteases which may be necessaryfor penetration of the basement membrane, in particular, tissueinhibitors of metalloproteinase TIMP-1 and TIMP-2; and other proteinssuch as protamine which has demonstrated angiostatic properties, decoyreceptors, drugs such as analogues of the angioinhibin fumagillin, e.g.,TNP-470 (Ingber et al., Nature 348:555-557 (1990), antibodies orantisense nucleic acid against angiogenic cytokines such as VEGF.Alternatively, the cells may be coupled to such angiogenesis inhibitingagent.

[0065] If the angiogenesis is associated with neoplastic growth the ECprogenitor cell may also be transfected with nucleic acid encoding, orcoupled to, anti-tumor agents or agents that enhance the immune system.Such agents include, for example, TNF, cytokines such as interleukin(IL) (e.g., IL-2, IL-4, IL-10, IL-12), interferons (IFN) (e.g.,IFN-.gamma.) and co-stimulatory factor (e.g., B7). Preferably, one woulduse a multivalent vector to deliver, for example, both TNF and IL-2simultaneously.

[0066] The nucleic acids are introduced into the cells by any method,which will result in the uptake and expression of the nucleic acid bythe cells. These can include vectors, liposomes, naked DNA,adjuvant-assisted DNA, catheters, gene gun, etc.

[0067] Vectors include chemical conjugates such as described in WO93/04701, which has targeting moiety (e.g. a ligand to a cellularsurface receptor), and a nucleic acid binding moiety (e.g. polylysine),viral vector (e.g. a DNA or RNA viral vector), fusion proteins such asdescribed in PCT/US 95/02140 (WO 95/22618) which is a fusion proteincontaining a target moiety (e.g. an antibody specific for a target cell)and a nucleic acid binding moiety (e.g. a protamine), plasmids, phage,etc. The vectors can be chromosomal, non-chromosomal or synthetic.

[0068] The vectors can be for example viral vectors, fusion proteins andchemical conjugates. Retroviral vectors include moloney murine leukemiaviruses and HIV-based viruses. One preferred HIV-based viral vectorcomprises at least two vectors wherein the gag and pol genes are from anHIV genome and the env gene is from another virus. DNA viral vectors arepreferred. These vectors include pox vectors such as orthopox or avipoxvectors, herpesvirus vectors such as a herpes simplex I virus (HSV)vector [Geller, A. I. et al., J. Neurochem, 64: 487 (1995); Lim, F., etal., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ.Press, Oxford England) (1995); Geller, A. I. et al., Proc Natl. Acad.Sci.: U.S.A.:90 7603 (1993); Geller, A. I., et al., Proc Nat. Acad. SciUSA: 87:1149 (1990)], Adenovirus Vectors [LeGal LaSalle et al., Science,259:988 (1993); Davidson, et al., Nat. Genet 3: 219 (1993); Yang, etal., J. Virol. 69: 2004 (1995)] and Adeno-associated Virus Vectors[Kaplitt, M. G., et al., Nat. Genet. 8:148 (1994)].

[0069] Pox viral vectors introduce the gene into the cells cytoplasm.Avipox virus vectors result in only a short term expression of thenucleic acid. Adenovirus vectors, adeno-associated virus vectors andherpes simplex virus (HSV) vectors are preferred for introducing thenucleic acid into neural cells. The adenovirus vector results in ashorter term expression (about 2 months) than adeno-associated virus(about 4 months), which in turn is shorter than HSV vectors. Theparticular vector chosen will depend upon the target cell and thecondition being treated. The introduction can be by standard techniques,e.g. infection, transfection, transduction or transformation. Examplesof modes of gene transfer include e.g., naked DNA, CaPO.sub.4precipitation, DEAE dextran, electroporation, protoplast fusion,lipofection, cell microinjection, viral vectors and use of the “genegun”.

[0070] To simplify the manipulation and handling of the nucleic acidencoding the protein, the nucleic acid is preferably inserted into acassette where it is preferably linked to a promoter. The promoter mustbe capable of driving expression of the protein in cells of the desiredtarget tissue. The selection of appropriate promoters can readily beaccomplished. Preferably, one would use a high expression promoter. Anexample of a suitable promoter is the 763-base-pair cytomegalovirus(CMV) promoter. The Rous sarcoma virus (RSV) (Davis, et al., Hum GeneTher 4:151 (1993)) and MMT promoters may also be used. Certain proteinscan express using their native promoter. Other elements that can enhanceexpression can also be included such as an enhancer or a system thatresults in high levels of expression such as a tat gene and tar element.This cassette can then be inserted into a vector, e.g., a plasmid vectorsuch as pUC118, pBR322, or other known plasmid vectors, that includes,for example, an E. coli origin of replication. See, Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratorypress, (1989). The plasmid vector may also include a selectable markersuch as the beta.-lactamase gene for ampicillin resistance, providedthat the marker polypeptide does not adversely effect the metabolism ofthe organism being treated. The cassette can also be bound to a nucleicacid binding moiety in a synthetic delivery system, such as the systemdisclosed in WO 95/22618.

[0071] If desired, the pre selected compound, e.g. a nucleic acid suchas DNA may also be used with a micro delivery vehicle such as cationicliposomes and adenoviral vectors. For a review of the procedures forliposome preparation, targeting and delivery of contents, see Mamino andGould-Fogerite, BioTechniques, 6:682 (1988). See also, Felgner and Holm,Bethesda Res. Lab. Focus, 11(2):21 (1989) and Maurer, R. A., BethesdaRes. Lab. Focus, 11(2):25 (1989).

[0072] Replication-defective recombinant adenoviral vectors can beproduced in accordance with known techniques. See, Quantin, et al.,Proc. Natl. Acad. Sci. USA, 89:2581-2584 (1992); Stratford-Perricadet,et al., J. Clin. Invest., 90:626-630 (1992); and Rosenfeld, et al.,Cell, 68:143-155 (1992).

[0073] The effective dose of the nucleic acid will be a function of theparticular expressed protein, the target tissue, the patient and his orher clinical condition. Effective amount of DNA are between about 1 and4000 mug, more preferably about 1000 and 2000, most preferably betweenabout 2000 and 4000.

[0074] Other delivery techniques can include 1) the electroporationtechnique and 2) the Adeno Associated Virus (AAV)

[0075] Alternatively, the cells for the tubularized tissue may be usedto inhibit angiogenesis and/or neoplastic growth by delivering to thesite of angiogenesis a cytotoxic moiety coupled to the cell. Thecytotoxic moiety may be a cytotoxic drug or an enzymatically activetoxin of bacterial, fungal or plant origin, or an enzymatically activepolypeptide chain or fragment (“A chain”) of such a toxin. Enzymaticallyactive toxins and fragments thereof are preferred and are exemplified bydiphtheria toxin A fragment, non-binding active fragments of diphtheriatoxin, exotoxin A (from Pseudomonas aeruginosa), ricin A chain, abrin Achain, modeccin A chain, alphasarcin, certain Aleurites fordii proteins,certain Dianthin proteins, Phytolacca americana proteins (PAP, PAPII andPAP-S), Momordica charantia inhibitor, curcin, crotin, Saponariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,and enomycini, Ricin A chain, Pseudomonas aeruginosa exotoxin A and PAPare preferred. Conjugates of the cells and such cytotoxic moieties maybe made using a variety of coupling agents. Examples of such reagentsare N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters such as dimethyladeipimidate HCl, active esters such as disuccinimidyl suberate,aldehydes such as glutaradehyde, bis-azido compounds such asbis(p-diazoniumbenzoyl)-ethylenediamine, diisocyanates such as tolylene2,6-diisocyante, and bis-active fluorine compounds such as1,5-difluoro-2,4-dinitrobenzene.

[0076] In another embodiment the scaffold is a filamentous scaffold:

[0077] The generation of a filamentous scaffold according to the presentinvention takes into account the following considerations: (i) Filamentsmust remain intact during cell adherence and growth thereupon, but atthe same time must rapidly erode within 20-30 days thereafter to obtaina functional blood vessel with a continuous lumen which supports bloodflow. (ii) Efficient adherence of cells to the filamentous scaffoldfollowed by cell proliferation around the filamentous scaffold to form acontinuous and uniform cell layer. Preferably, the filamentous scaffoldof the present invention is a solid scaffold capable of supporting cellgrowth thereupon. Such a scaffold can mimic a blood vessel lumen andform a blood vessel having even small capillary diameter of 5-50microns. (iii) The filamentous scaffold must be strong and flexibleenough to allow formation of flexible thin filaments having a diameterranging between 5-500 microns.

[0078] Various types of biodegradable polymers meet these criteria,including, for example, thin cellulose fibers. Cellulose fibbers can bemodified by oxidation with, for example, periodate in aqueous medium,rendering the fibers more susceptible to hydrolytic degradation(biodegradation). The degree of oxidation determines the strength of thefiber and its degradation profile. These oxidized fibers can be furthermodified by impregnation with a biodegradable polymer such aspoly(lactide-glycolide) so as to be more susceptible to biologicaldegradation. Optionally, fiber aldehyde groups can be reacted with aminocontaining hydrophilic or hydrophobic safe molecules including aminoacids.

[0079] Alternatively, polymers and copolymers based on hydroxy alkylacid polyesters, polyphosphazene, poly(carbonates) and poly(phosphateesters), can also be used. Polymers based on lactide and glycolide acidsare better suited for use with the filamentous scaffold of the presentinvention since it has been previously shown that such materials arecapable of supporting cell growth and can be safely transplanted inhumans (Shand and Heggie 2000).

[0080] These polymers can also be modified to meet the requirementsdescribed above. For example, block and random copolymers of lactideacid and glycolic acid having a molecular weight greater than 10,000,can be spun into thin filaments. To prevent accelerated erosion whenexposed to an environment enriched with degradative enzymes, copolymersincluding 30 to 70% lactic acid may be used to delay degradation to afew weeks post transplantation.

[0081] Increased flexibility of the filaments can be obtained by addingplasticizing agents such as, for example, tributyl citrate, tributylcitrate acetate, phospholipids, oleate esters and the like to thepolymer blend or by incorporating agents, such as, for example,ricinoleic acid into the polymer chain.

[0082] The mechanical properties of the filamentous scaffold must bemaximized when supporting formation of a blood vessel such as an artery,which has to exhibit resistance to high blood pressure. This can beachieved by various cross-linking methods, interlinking the filamentouspolymers, described herein above.

[0083] Preferably, cross-linking is achieved via stereocomplexationwhich utilizes stereoisomers, such as the stereoisomers of copolymer oflactic acid, as linker molecules for stereo-cross-linking the polymerbackbone (see Example 1 of the Examples section for further detail).

[0084] Continuous scaffold:

[0085] The continuous scaffold of the invention is designed so as tosupport tissue formation in and around the filamentous scaffold. Thecontinuous scaffold can be composed of any of the polymers describedhereinabove and/or any other polymers suitable for supporting structuraltissue colonization/proliferation.

[0086] For example, polysaccharide such as cross-linked dextran,arabinogalactan, chitosan, polyactide-glycolide, alginates, pullulan,hyaluronic acid, and the like, and proteins such as gelatine, collagen,fibrin, fibrinogen, albumin, and the like, can be is used to form acontinuous (cross linked) scaffold with a predetermined pore size.Alternatively, synthetic polymers such as, lactide and glycolide foamscan also be used.

[0087] Preferably, the continuous scaffold component is generated undermild conditions. This enables to form the continuous scaffold componentover a filamentous scaffold component, which is already seeded withcells. Compositions based on viscous hyaluronic acid solutions,alginates cross-linked by calcium salts and proteins cross-linked bydenaturation or non-harmful molecules can be used to form the continuousscaffold component over an already seeded filamentous scaffoldcomponent.

[0088] Alternatively, stereocomplexed hydrophilic polymers including,natural polysaccharides, proteins, and polymers based on ethylene andpropylene glycol and mixtures thereof can also be used.

[0089] In another embodiment biodegradable polymeric scaffold isnon-toxic and bioabsorbable when introduced into a living organism andany degradation products of the biopolymer should also be non-toxic tothe organism.

[0090] The source of molecules, which form biopolymers, include mammalssuch as pigs, e.g., near-term fetal pigs, sheep, fetal sheep, cows, andfetal cows. Other sources of the molecules, which can form biopolymers,include both land and marine vertebrates and invertebrates. In oneembodiment, the collagen can be obtained from skins of near-term,domestic porcine fetuses, which are harvested intact, enclosed in theiramniotic membranes. Collagen or combinations of collagen types can beused in the matt and matt compositions described herein. Examples ofcollagen or combinations of collagen types include collagen type I,collagen type II, collagen type III, collagen type IV, collagen type V,collagen type VI, collagen type VII, collagen type VIII, collagen typeIX, collagen type X, collagen type XI, collagen type XII, collagen typeXIII, and collagen type XIV. A preferred combination of collagen typesincludes collagen type I, collagen type III, and collagen type IV.Preferred mammalian tissues from which to extract the molecules, whichcan form biopolymer, include entire mammalian fetuses, e.g., porcinefetuses, dermis, tendon, muscle and connective tissue. As a source ofcollagen, fetal tissues are advantageous because the collagen in thefetal issues is not as heavily cross-linked as in adult tissues. Thus,when the collagen is extracted using acid extraction, a greaterpercentage of intact collagen molecules is obtained from fetal tissuesin comparison to adult tissues. Fetal tissues also include variousmolecular factors, which are present in normal tissue at differentstages of animal development.

[0091] In another embodiment, the scaffold of the invention can supportnew vascular tissue or tubularized tissue formation by serving as atemplate to the development of a tissue in the direction outlined by thescaffold.

[0092] In another embodiment, the tissue is formed ex vivo and thetransplantation into the human body is conducted the entodelial cellswill sprout form new blood vessels which direction will be paved by thefilament that will slowly degrade.

[0093] In another embodiment, the scaffold is a biodegradable scaffoldand is degraded upon being introduced to the body, so as to permits alumen in which nutrients, excretions and gases can pass through. Thescaffold is degradable upon exposure to predetermined environmentalconditions such as the conditions exist in the body, namely, hydrolyticenzymes, presence of a low pH, namely pH with is less than 5 andreducing conditions.

[0094] In another embodiment, the scaffold comprises thermo-regulatedpolymers that change mechanical properties as a result to changes intemperature thus allowing degradation (liquified state) to becontrolled.

[0095] In another embodiment of the invention, the scaffold furthercomprising macromolecules necessary for cell growth, morphogenesis,differentiation, and tissue building can also be added to the biopolymermolecules or to the biopolymer fibrils to further promote cell in growthand tissue development and organization of the cells. The term“macromolecules necessary for cell growth, morphogenesis,differentiation, and tissue building” refers to those molecules, e.g.,macromolecules such as proteins, which participate in the development oftissue. Such molecules contain biological, physiological, and structuralinformation for development or regeneration of the tissue structure andfunction. Examples of these macromolecules include, but are not limitedto, growth factors, extracellular matrix proteins, proteoglycans,glycosaminoglycans and polysaccharides. Alternatively, the biopolymermatts, matt composites, and matt compositions of the invention caninclude extracellular matrix macromolecules in particulate form orextracellular matrix molecules deposited by cells or viable cells.

[0096] The term “growth factors” is art recognized and is intended toinclude, but is not limited to, one or more of platelet derived growthfactors (PDGF), e.g., PDGF AA, PDGF BB; insulin-like growth factors(IGF), e.g., IGF-I, IGF-II; fibroblast growth factors (FGF), e.g.,acidic FGF, basic FGF, beta.-endothelial cell growth factor, FGF 4, FGF5, FGF 6, FGF 7, FGF 8, and FGF 9; transforming growth factors (TGF),e.g., TGF-P1, TGF-beta.1.2, TGF-beta 2, TGF-beta 3, TGF-beta 5; bonemorphogenic proteins (BMP), e.g., BMP 1, BMP 2, BMP 3, BMP 4; vascularendothelial growth factors (VEGF), e.g., VEGF, placenta growth factor;epidermal growth factors (EGF), e.g., EGF, amphiregulin, betacellulin,heparin binding EGF; interleukins, e.g., IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14; colonystimulating factors (CSF), e.g., CSF-G, CSF-GM, CSF-M; nerve growthfactor (NGF); stem cell factor; hepatocyte growth factor, and ciliaryneurotrophic factor. The term encompasses presently unknown growthfactors that may be discovered in the future, since theircharacterization, as a growth factor will be readily determinable bypersons skilled in the art.

[0097] In another embodiment the scaffold comprises a therapeutic agent.Those can be selected for example without being limited anti-infective,a hormone, an analgesic, an anti-inflammatory agent, a chemotherapeuticagent, an anti-rejection agent, a prostaglandin, RGD peptide andcombinations thereof.

[0098] In another embodiment, the invention provides a method ofenhancing blood vessel formation in a patient in need thereof,comprising the following steps: selecting the patient in need thereof;isolating a cell from the patient; obtaining a scaffold; seeding saidcell exterior to said scaffold so as to obtain a scaffold encircled bycells; growing the scaffold encircled by the cells so as to form atubularized tissue with the scaffold contained by, so as to support thetubularized tissue; thereby obtaining a tubularized tissue; andre-administering the tubularized tissue to the patient in need thereof,thereby enhancing blood vessel formation in a patient in need thereof.

[0099] As was shown in the Examples section, Endothelial b-End-2 cellswere cultured on polymeric filamentous scaffolds for 7 and 14 days in arotary bioreactor. It was observed that endothelial monolayer has coatedall the surface area of the polymeric filament after 7 days of culturing(FIG. 4a). The coated cells had the morphology of flat endotheliumlining the surface of the scaffold. The vascular tissue formed ex vivowas transplanted subcutaneous into nude mice. 28, 42, 56, and 70 daysafter transplantation mice were sacrificed and samples were analysedmicroscopically. Analysis revealed the beginning of maturation of theprimitive vessels allowing the flow of red blood cells inside theforming lumen of the vessels as the polymer degrades (FIG. 7b).Moreover, Reticulum stainings have shown the formation of basementmembrane, which is typical for blood vessels, surrounding the engineeredvessels (FIGS. 7c &7 d). Analysis of the cells revealed that theengineered vessels were surrounded by GFP positive cells (FIGS. 1e, 6 b& 6 c) having the morphology of endothelial cells and expressing PECAM(FIG. 10). This observation indicates that engrafted endothelial cellsdifferentiated into mature endothelium and that the engineered vesselswere undergoing maturation in vivo and acquired functionality.

[0100] In another embodiment the cell is provided from a different i.e.universal donor, or in another embodiment, the cell is derived from adifferent animal and is than seeded exterior to the scaffold so as toobtain a scaffold encircled by cells; the next steps are growing thescaffold encircled by the cells so as to form a tubularized tissue withthe scaffold contained by, so as to support the tubularized tissue;thereby obtaining a tubularized tissue; and administering thetubularized tissue to the patient in need thereof, thereby enhancingblood vessel formation in a patient in need thereof.

[0101] Post-natal neovascularization is believed to result exclusivelyfrom the proliferation, migration, and remodeling of fullydifferentiated endothelial cells (ECs) derived from pre-existing nativeblood vessels. This adult paradigm, referred to as angiogenesis,contrasts with vasculogenesis, the term applied to Formation ofembryonic blood vessels from EC progenitors. In contrast toangiogenesis, vasculogenesis typically begins as a cluster formation, orblood island, comprised of EC progenitors (e.g. angioblasts) at theperiphery and hematopoietic stem cells (HSCs) at the center (3). Inaddition to this intimate and predictable spatial association, such ECprogenitors and HSCs share certain common antigenic determinants,including flk-1, tie-2, and CD-34. Consequently, these progenitor cellshave been interpreted to derive from a common hypothetical precursor,the hemangioblast.

[0102] In accordance with the invention, the tubularized tissue can beused in a method for regulating angiogenesis, i.e., enhancing orinhibiting blood vessel formation, in a selected patient. For example,the tubularized tissue can be used to enhance angiogenesis or to deliveran angiogenesis modulator, e.g. anti- or pro-angiogenic agents,respectively to sites of pathologic or utilitarian angiogenesis.

[0103] If it is desirable to further enhance angiogenesis, endothelialcell mitogens may also be administered to the patient in conjunctionwith, or subsequent to, the administration of the EC progenitor cells.Endothelial cell mitogens can be administered directly, e.g.,intra-alterially, intramuscularly, or intravenously, or nucleic acidencoding the mitogen may be used. See, Baffour, et al., supra (bFGF);Pu, et al, Circulation, 88:208-215 (1993) (aFGF); Yaniagisawa-Miwa, etal., supra (bFGF); Ferrara, et al., Biochem. Biophys. Res. Commun.,161:851-855 (1989) (VEGF); (Talkeshita, et al., Circulation, 90:228-234(1994)).

[0104] Additionally, in another embodiment, tubularized tissue can beused to induce re-endothelialization of an injured blood vessel, andthus reduce restenosis by indirectly inhibiting smooth muscle cellproliferation.

[0105] In another embodiment the tubularized tissue can be used toregulate vasculogenesis For example, the tubularized tissue can be usedto enhance vasculogenesis or to deliver an vasculogenesis modulator,e.g. anti- or pro-vasculogenic agents, respectively to sites ofpathologic or utilitarian vasculogenesis.

[0106] In another embodiment the tubularized tissue can be used in amethod of treating hypoxia, avascular necrosis, foot ulcer or gangreneresult from diabetes, athersclerosis, stroke, wound, fracture of bone,comprising administering to the patient host an effective amount of thetubularized tissue of the invention so as to increasing the vascularityof a tissue thereby treating hypoxia, avascular necrosis, foot ulcer organgrene result from diabetes, athersclerosis, stroke, wound, fractureof bone.

[0107] In one embodiment, the present invention may be used to enhanceblood vessel formation in ischemic tissue, i.e., a tissue having adeficiency in blood as the result of an ischemic disease. Such tissuescan include, for example, muscle, brain, kidney and lung. Ischemicdiseases include, for example, cerebrovascular ischemia, renal ischemia,pulmonary ischemia, limb ischemia, ischemic cardiomyopathy andmyocardial ischemia.

[0108] The tubularized tissue can be used for transplantation intoorgan/tissue in need for increased vascularity such as regeneratingtissue, tissue suffering from avascular necrosis or hypoxia. For exampleischemic cardiac tissue can be improved by transplantation of engineeredcapillary network. In another example such a “patch” of engineeredcapillaries can be implanted into a regenerating wound site as skinwounds or bone fractures etc.

[0109] In another embodiment single vessels can be used for bypassingoccluded vessels as in cardiac coronary artery bypasses. In thisparticular situation, the present concept present a specific advantageby “protecting” the lumen of the vessel by the polymer and thereforepreventing collapse of the bypass engineered vessel.

[0110] One of the major problems of bioartificial implants is the needfor a well-branched vascular network, which can provide the engineeredtissue with continuous supply of oxygen and nutrients at thetransplantation site. To date all attempts to address this issue haveresulted in poor vascularization of engrafted tissues

[0111] The tubularised tissue can be used also for the promoting theintegration of grafts, tissues and organs transplantation. Adding thevascular tissue to the graft will promote angiogenesis andvascularization of the graft and can increase the integration. Forexample transplantation of a “patch” of engineered capillary networksfollowed by skin tissue grafts can promote the successful integration ofthe skin grafts. In another embodiment, a network of blood vessels canbe formed in order to ex-vivo growing other cell culture or tissueswhich will be supplied with gases and nutrients from the blood network.

[0112] Promotion of vascularization is achieved in this concept by theintegration of the engineered tissue in vivo with the host vasculartissue. This process is achieved by angiogenesis invoked from theengrafted vascular tissue and from the host vascular tissue enablingboth integration of the engineered vascular tissue to the host vascularsystem and promoting increased collateral vessels and anastemoses (seediagram). In order to effectively influence this process to promoteangiogenesis, integration and stabilisation of new vessels growth factordelivery can be used. Time dependent delivery of VEGF (Angiogenesis) andAng-1 (stabilisation) by means of protein or gene delivery can be usedfor this purpose (see diagram).

[0113] In another embodiment, a nucleic acid encoding the EC mitogen canbe administered to a blood vessel perfusing the ischemic tissue or to asite of vascular injury via a catheter, for example, a hydrogelcatheter, as described by U.S. Ser. No. 08/675,523, the disclosure ofwhich is herein incorporated by reference. The nucleic acid also can bedelivered by injection directly into the ischemic tissue using themethod described in U.S. Ser. No. 08/545,998.

[0114] As used herein the term “endothelial cell mitogen” means anyprotein, polypeptide, mutein or portion that is capable of, directly orindirectly, inducing endothelial cell growth. Such proteins include, forexample, acidic and basic fibroblast growth factors (aFGF and bFGF),vascular endothelial growth factor (VEGF), epidermal growth factor(EGF), transforming growth factor alpha and beta. (TGF-alpha. andTFG-beta.) platelet-derived endothelial growth factor (PD-ECGF),platelet-derived growth factor (PDGF), tumor necrosis factor a(TNF-alpha), hepatocyte growth factor (HGF), insulin like growth factor(IGF), erythropoietin, colony stimulating factor (CSF), macrophage-CSF(M-CSF), granulocyte/macrophage CSF (GM-CSF) and nitric oxidesynthase(NOS). See, Klagsbrun, et al., Annu. Rev. Physiol., 53:217-239 (1991);Folkman, et al., J. Biol., Chem., 267:10931-10934 (1992) and Symes, etal., Current Opinion in Lipidology, 5:305-312 (1994). Muteins orfragments of a nitrogen may be used as long as they induce or promote ECcell growth.

[0115] In one embodiment the endothelial cell mitogen contains asecretory signal sequence that facilitates secretion of the protein.Proteins having native signal sequences, e.g., VEGF can be used.Proteins that do not have native signal sequences, e.g., bFGF, can bemodified to contain such sequences using routine genetic manipulationtechniques. See, Nabel et al., Nature, 362:844 (1993).

[0116] The nucleotide sequence of numerous endothelial cell mitogens,are readily available through a number of computer databases, forexample, GenBank, EMBL and Swiss-Prot. Using this information, a DNAsegment encoding the desired may be chemically synthesized or,alternatively, such a DNA segment may be obtained using routineprocedures in the art, e.g, PCR amplification. A DNA encoding VEGF isdisclosed in U.S. Pat. No. 5,332,671, the disclosure of which is hereinincorporated by reference.

[0117] In certain situations, it may be desirable to use nucleic acidsencoding two or more different proteins in order optimize thetherapeutic outcome. For example, DNA encoding two proteins, e.g., VEGFand bFGF, call be used, and provides an improvement over the use of bFGFalone. In another embodiment an angiogenic factor can be combined withother genes or their encoded gene products to enhance the activity oftargeted cells, while simultaneously inducing angiogenesis, including,for example, nitric oxide synthase, L-arginine, fibronectin, urokinase,plasminogen activator and heparin.

[0118] The term “effective amount” means a sufficient amount ofcompound, e.g. nucleic acid delivered to produce an adequate level ofthe endothelial cell mitogen, i.e., levels capable of inducingendothelial cell growth and/or inducing angiogenesis. Thus, theimportant aspect is the level of mitogen expressed. Accordingly, one canuse multiple transcripts or one can have the gene under the control of apromoter that will result in high levels of expression. In analternative embodiment, the gene would be under the control of a factorthat results ill extremely high levels of expression, e.g., tat and thecorresponding tar element.

[0119] The tubularized tissue obtained in the invention can be servedfor determining cellular or tissue function of an agent such as itseffect on processes such as proliferation, apoptosis, toxicityangiogenesis, vasculogenesis or differentiation comprising the followingsteps: obtaining the tubularized tissue described in the invention;contacting the tubularized tissue with an agent, so as to obtain atubularized tissue contacted with an agent; comparing cellular or tissuefunction of the tubularized tissue contacted with an agent, to atubularized tissue which was not contacted with the agent, therebydetermining cellular or tissue function of the agent. The agent can be aprotein, a hormone, a RNA molecule, a DNA molecule, an antibody anantagonist and the like.

[0120] In another embodiment, the tubularized tissue can be served forscreening candidate genes which are involved in cellular or tissuefunction such as proliferation, apoptosis, toxicity angiogenesis,vasculogenesis or differentiation, the method comprising the steps of:obtaining the tubularized tissue; obtaining mRNA from the tubularizedtissue and the cell; synthesizing cDNA from them RNA; amplifying thecDNA-hybrid, so as to obtain an amplified product; detecting theamplified product; and comparing the amplified products from thetubularized tissue and the cell, thereby identifying candidate nucleicacid sequence which is involved in the cellular or tissue function.

[0121] This Ex vivo system can be utilized for evaluating the effects ofphysical/biomechanical effects on tissue formation and maturation exvivo and to replace the controversial animal models.

EXAMPLES Experimental Procedures

[0122] Tissue Culture:

[0123] cells were cultured in low glucose, low bicarbonate DMEM medium(Beit Haemek)+10% fetal calf serum (Biet Haemek), the environmentalconditions were of 5% CO2 and 37 0 C.

[0124] Seeding of Filament-Like Polymeric Scaffold with VascularEndothelial Cells:

[0125] The filamentous scaffold designed for the vascular tissue wasseeded with to endothelial cells, b-End-2 (Jia, G Q et. al., 1999 andHahne M et. al., 1993), genetically engineered to express the reportergene, green fluorescent protein (GFP), 2 million cells per scaffold(calculated cells per cm2 polymeric scaffold surface) in a stirringflask for 24 hrs (Zipori 1989, Jia et al. 1999, Vunjak-Novakovic et al.2000). Culture medium contains DMBM supplemented with 10% FCS, highglucose.

[0126] Complete Endothelialization of the Filament-Like PolymericScaffold:

[0127] In order to achieve a spatially uniform distribution of theendothelial cells, to promote rapid, homogeneous tissue development, anda confluent cell monolayer covering the scaffold's surface, the seededscaffold was cultured in a rotary bioreactor for 7 days

[0128] Ex vivo Analysis of Endothelialized Filament-Like PolymericScaffold Cultured in a Rotary Bioreactor:

[0129] Following 7 days of culturing, scaffold was harvested and fixedusing three different methods each for a different analysis:

[0130] Protein Localization:

[0131] PECAM (CD31): Serial sections (5-6 μm) of PFA-fixed,paraffin-embedded tissue were deparaffinized in xylene and rehydratedthrough graded ethanols. Following antigen retrieval (incubation incitrate buffer and heating) and inhibition of endogenous peroxidase(incubation in 1% hydrogen peroxide in TBS) immunostaining was performedusing mouse monoclonal antibody directed against PECAM which was used asan endothelial cell marker (PharMingen, San Diego, Calif.). Sectionswere incubated with avidin-biotin-peroxidase complex. Antibody bindingwas detected by DAB (Shweiki D et al. 1992).

[0132] GFP: polyclonal rabbit anti-GFP (Clontech Laboratories, PaloAlto, Calif.) was used for GFP immunostaining as a gene reporter markerfor b-End-2 endothelial cell line (FIG. 1e). A second method for GFPlocalization study was used included PFA-fixed OCT embedded tissue usingfluorescence microscopy (FIGS. 6b & 6 c). Reticulum staining(Wilder-silver nitrate stain) specific histochemical staining forbasement membrane.

[0133] In vivo Transplantation:

[0134] Mice were anaesthesized using 30 ml of ketamine-Xylazine mixture(85%, 15% respectively) injected intra peritoneal per mouse. Skin wasshaved in the low back area of the mouse, and swabbed with chlorhexidinesolution 0.5%. Skin was cut with scissors, and subcutaneous pocket wasformed in the sacral area. Endothelialized filament-like polymericscaffold was placed in the subcutaneous pocket, covered with the skin,which was sutured with clips. The skin and the clips were swabbed withchlorhexidine.

[0135] In vivo Analysis of Engrafted Vascular Tissue:

[0136] MRI Analysis of Blood Vessel Density, Functionality andMaturation:

[0137] MRI experiments were performed on a horizontal 4.7 T BrikerBiospec spectrometer, using an actively RF decoupled surface coil, 2 cmin diameter, and a birdcage transmission coil (Abramovitch et al.,1999). Mice were anesthetized (75 mg/kg Ketamine+3 mg/kg Xylazine, i.p.)and placed supine with the tumor located at the center of the surfacecoil. Transplanted primitive micro-capillary network tissue'svascularity was reflected by reduction of the mean signal intensityinside the tumor in gradient echo T2* weighted images (TR-repetitiontime=100 ms; TF-echo time=10 ms). Data is reported as the apparentvessel density {AVD=−ln(s(t)/s(0))}, in which s(t) is the mean intensityin the transplanted primitive micro-capillary network tissue, and s(0)is the mean intensity of a distant muscle, as described (Abramovitch etal., 1998). Functionality and maturation of the neovasculature weredetermined from gradient echo images acquired during the inhalation ofair, air—CO2 (95% air and 5% CO2), and oxygen-CO2 (95% oxygen and 5%CO2; carbogen), as described (Abramovitch et al., 1999). Four imageswere acquired at each gas mixture (65 s/image; slice thickness=0.6 mm;TR=100 ms; TE=10 ms; spectral width=25,000 Hz; field of view=3 cm;256×128 pixels; in plane resolution=110 □m; four averages). Otherexperimental details were as reported previously (Abramovitch et al.,1999).

[0138] Data Analysis. MRI data were analyzed on a PC computer using IDLsoftware (Research Systems Inc.). Vascular function (VF) was derivedfrom images acquired during inhalation of carbogen (95% oxygen 5% CO2)and air—CO2 (95% air 5% CO2) as follows:${VF} = {{b\quad \Delta \quad Y} = \frac{\ln \left( {I_{carbogen}/I_{{air} - {CO}_{2}}} \right)}{{TE}*C_{MRI}}}$

[0139] Where TE is the echo time, Y is the fraction of oxy-hemoglobin, bis the volume fraction of blood and CMRI=599 s−1 at 4.7 T. Thisparameter measures the capacity of oxygen delivery from the lungs toeach pixel in the image (Abramovitch et al., 1998). Vasodilation (VD)was derived from air and air-CO2 images, as described (Abramovitch etal., 1999; Abramovitch et al., 1998). Positive VD corresponds toincreased signal intensity by hypercapnia, due to elevated bloodoxygenation and/or increased blood flow (Abramovitch et al., 1998). Dataare presented in color maps overlayed on the original baseline image forabsolute values of VD and VF>0.005 (Abramovitch et al., 1999).

Experimental Results

[0140] Ex-Vivo Formation of Primitive Vascular Tissue:

[0141] Endothelial b-end-2 cells were cultured on polymeric filamentousscaffolds for 7 and 14 days in a rotary bioreactor. It was observed thatendothelial monolayer has 5 coated all the surface area of the polymericfilament after 7 days of culturing (FIG. 4a). The coated cells had themorphology of flat endothelium lining the surface of the scaffold.

[0142] Primitive Vascular Tissue Formed ex vivo, Mature and Function invivo:

[0143] Primitive capillary network formed ex vivo was transplantedsubcutaneous into nude mice. 28, 42, 56, and 70 days aftertransplantation mice were sacrificed and samples were analysedmicroscopically. Analysis revealed the beginning of maturation of theprimitive vessels allowing the flow of red blood cells inside theforming lumien of the vessels as the polymer degrades (FIG. 7b).Moreover, Reticulum staining has showed the formation of basementmembrane, which is typical for blood vessels, surrounding the engineeredvessels (FIGS. 7c &7 d). Analysis of the cells revealed that theengineered vessels were surrounded by GFP positive cells (FIGS. 1e, 6 b& 6 c) having the morphology of endothelial cells and expressing PECAM(FIG. 10). This observation indicates that engrafted endothelial cellsdifferentiated into mature endothelium and that the engineered vesselswere undergoing maturation in vivo and acquired functionality.

What is claimed is:
 1. A tubularized tissue made according to the stepsof: seeding a cell on the exterior surface of scaffold so as to obtain ascaffold encircled by cells; incubating said scaffold encircled by saidcells so as to form a tubularized tissue; thereby obtaining atubularized tissue.
 2. The tubularized tissue of claim 1, whereby in thestep of obtaining a tubularized tissue, the tubularized tissue is amonolayer tubular tissue.
 3. The tubularized tissue of claim 1, wherebyin the step of obtaining a tubularized tissue, the tubularized tissue isa multi-layer tubular tissue.
 4. The tubularized tissue of claim 1,whereby in the step of obtaining a tubularized tissue, the tubularizedtissue is a multi-layer tubularized tissue comprising more than one typeof cells.
 5. The tubularized tissue of claim 1, whereby in the step ofobtaining a tubularized tissue, said tubularized tissue is avascularized tissue.
 6. The tubularized tissue of claim 1, whereby inthe step of seeding a cell on the exterior surface of scaffold, saidscaffold is in the form of a string.
 7. The tubularized tissue of claim1, whereby in the step of seeding a cell on the exterior surface ofscaffold, said scaffold is a biodegradable scaffold.
 8. The tubularizedtissue of claim 2, wherein the biodegradable scaffold is a polymericbiodegradable scaffold.
 9. The tubularized tissue of claim 2, whereinthe polymeric biodegradable scaffold comprising cross-linked dextran,arabinogalactan, chitosan, polyactide-glycolide, alginates, pullulan,gelatin, collagen, fibrin, fibrinogen or albumin.
 10. The tubularizedtissue of claim 1, whereby in the step of obtaining a scaffold, thediameter of said scaffold is 4-500 um.
 11. The tubularized tissue ofclaim 1, whereby in the step of obtaining a scaffold, said scaffold isdegradable upon exposure to predetermined environmental conditions. 12.The tubularized tissue of claim 1, whereby in the step of obtaining ascaffold, said scaffold is a non-biodegradable scaffold.
 13. Thetubularized tissue of claim 1, whereby in the step of obtaining ascaffold, said scaffold comprises macromolecules necessary for cellgrowth, morphogenesis, differentiation, or tissue building andcombinations thereof.
 14. The tubularized tissue of claim 13, whereinthe macromolecules necessary for cell growth is a bone morphogenicprotein, a bone morphogenic-like protein, an epidermal growth factor, afibroblast growth factor, a platelet derived growth factor, an insulinlike growth factor, a transforming growth factor, a vascular endothelialgrowth factor, Ang1, PIGF and combinations thereof.
 15. The tubularizedtissue of claim 1, whereby in the step of obtaining a scaffold, saidscaffold contains also a therapeutic agent.
 16. The tubularized tissueof claim 15, wherein said therapeutic agent is an anti-infective, ahormone, an analgesic, an anti-inflammatory agent, a chemotherapeuticagent, an anti-rejection agent, a prostaglandin, RGD peptide andcombinations thereof.
 17. The tubularized tissue of claim 15, whereinsaid therapeutic agent is a nucleic acid sequence which encodes ananti-infective, a hormone, an analgesic, an anti-inflammatory agent, achemotherapeutic agent, an anti-rejection agent, a prostaglandin, RGDpeptide and combinations thereof.
 18. The tubularized tissue of claim 1,wherein said cell is engineered to express a therapeutic agent and/or amacromolecule necessary for cell growth, morphogenesis, differentiation,or tissue building and combinations thereof.
 19. The tubularized tissueof claim 18, wherein said therapeutic agent is anti-infective, ahormone, an analgesic, an anti-inflammatory agent, a chemotherapeuticagent, an anti-rejection agent, a prostaglandin, RGD peptide andcombinations thereof.
 20. The tubularized tissue of claim 18, whereinsaid macromolecule necessary for cell growth, morphogenesis,differentiation, or tissue building and combinations thereof is a bonemorphogenic protein, a bone morphogenic-like protein, an epidermalgrowth factor, a fibroblast growth factor, a platelet derived growthfactor, an insulin like growth factor, a transforming growth factor, avascular endothelial growth factor, Ang1, PIGF and combinations thereof.21. The tubularized tissue of claim 1, whereby in the step of obtaininga cell, said cell is a mesenchymal stem cell, mesodermal progenitorcell, endothelial precursor cell or neonatal dermal micro vascularendothelial cells.
 22. The tubularized tissue of claim 1, whereby in thestep of obtaining a cell, said cell is capable of differentiating intoan endotelial cell.
 23. The tubularized tissue of claim 1, whereby inthe step of obtaining a cell, said cell is isolated from peripheralblood.
 24. The tubularized tissue of claim 1, whereby in the step ofobtaining a cell, said cell is isolated from mammalian artery, vein orcapillary.
 25. The tubularized tissue of claim 1, whereby in the step ofobtaining a cell said cell is genetically engineered to express areporter gene.
 26. The tubularized tissue of claim 25, wherein thereporter gene is a fluorescent protein, Luciferase or b-gal.
 27. Thetubularized tissue of claim 1, wherein said step of growing scaffoldencircled by cells so as to form a tubularized tissue is by the use of abioreactor.
 28. The tubularized tissue of claim 1, wherein thetubularized tissue is a single vessel, composition of several vessels ora capillary network.
 29. A method of maturing cells into a tubularizedtissue comprising the following steps: seeding a cell on the exteriorsurface of scaffold so as to obtain a scaffold encircled by cells;incubating said scaffold encircled by said cells so as to form atubularized tissue; thereby maturing cells into a tubularized tissue.30. The method of claim 29, wherein said tubularized tissue is amonolayer tubular tissue.
 31. The method of claim 29, wherein saidtubularized tissue is a multilayer tubular tissue.
 32. The method ofclaim 29, wherein said tubularized tissue is a multi-layer tubularizedtissue comprising more than one type of cells.
 33. The method of claim29, wherein said tubularized tissue is a vascular tissue.
 34. The methodof claim 29, wherein said scaffold is in the form of a string.
 35. Themethod of claim 29, wherein said scaffold is a biodegradable scaffold.36. The method of claim 35, wherein said biodegradable scaffold is apolymeric biodegradable scaffold.
 37. The method of claim 36, whereinsaid polymeric biodegradable scaffold comprising cross-linked dextran,arabinogalactan, chitosan, polyactide-glycolide, alginates, pullulan,gelatin, collagen, fibrin, fibrinogen or albumin.
 38. The method ofclaim 29, wherein the diameter of said scaffold is 4-500 um.
 39. Themethod of claim 29, wherein said scaffold is degradable upon exposure topredetermined environmental conditions.
 40. The method of claim 29,wherein said scaffold is a non-biodegradable scaffold.
 41. The method ofclaim 29, wherein said scaffold comprises macromolecules necessary forcell growth, morphogenesis, differentiation, or tissue building andcombinations thereof.
 42. The method of claim 41, wherein saidmacromolecules necessary for cell growth is a bone morphogenic protein,a bone morphogenic-like protein, an epidermal growth factor, afibroblast growth factor, a platelet derived growth factor, an insulinlike growth factor, a transforming growth factor, a vascular endothelialgrowth factor, Ang1, PIGF and combinations thereof.
 43. The method ofclaim 29, wherein said scaffold contains also a therapeutic agent. 44.The method of claim 43, wherein said therapeutic agent is ananti-infective, a hormone, an analgesic, an anti-inflammatory agent, achemotherapeutic agent, an anti-rejection agent, a prostaglandin, RGDpeptide and combinations thereof.
 45. The method of claim 29, whereinsaid cell is engineered to express a therapeutic agent and/or amacromolecule necessary for cell growth, morphogenesis, differentiation,or tissue building and combinations thereof.
 46. The method of claim 45,wherein said therapeutic agent is anti-infective, a hormone, ananalgesic, an anti-inflammatory agent, a chemotherapeutic agent, ananti-rejection agent, a prostaglandin, RGD peptide and combinationsthereof.
 47. The method of claim 45, wherein said macromoleculenecessary for cell growth, morphogenesis, differentiation, or tissuebuilding and combinations thereof is a bone morphogenic protein, a bonemorphogenic-like protein, an epidermal growth factor, a fibroblastgrowth factor, a platelet derived growth factor, an insulin like growthfactor, a transforming growth factor, a vascular endothelial growthfactor, Ang1, PIGF and combinations thereof.
 48. The method of claim 29,wherein said cell is a mesenchymal stem cell, mesodermal progenitorcell, endothelial precursor cell or neonatal dermal micro vascularendothelial cells.
 49. The method of claim 29, wherein said cell iscapable of differentiating into an endotelial cell.
 50. The method ofclaim 29, wherein said cell is isolated from peripheral blood.
 51. Themethod of claim 29, wherein said cell is isolated from mammalian artery,vein or capillary.
 52. The method of claim 29, wherein said cell isgenetically engineered to express a reporter gene.
 53. The method ofclaim 52, wherein the reporter gene is a fluorescent protein, Luciferaseor b-gal.
 54. The method of claim 29, wherein said step of growingscaffold encircled by cells so as to form a tubularized tissue is by theuse of a bioreactor.
 55. The method of claim 29, wherein tubularizedtissue is a single vessel, composition of several vessels or a capillarynetwork.
 56. A method of enhancing blood vessel formation in a patientin need thereof, comprising the following steps: selecting the patientin need thereof; isolating a cell from the patient; seeding a cell onthe exterior surface of scaffold so as to obtain a scaffold encircled bycells; incubating said scaffold encircled by said cells so as to form atubularized tissue; and readministering said tubularized tissue to thepatient in need thereof, thereby enhancing blood vessel formation in apatient in need thereof.
 57. The method of claim 56, wherein saidtubularized tissue is a monolayer tubular tissue.
 58. The method ofclaim 56, wherein said tubularized tissue is a multi-layer tubulartissue.
 59. The method of claim 56, wherein said tubularized tissue is amulti-layer tubularized tissue comprising more than one type of cells.60. The method of claim 56, wherein said tubularized tissue is avascular tissue.
 61. The method of claim 56, wherein said scaffold is inthe form of a string.
 62. The method of claim 56, wherein said scaffoldis a biodegradable scaffold.
 63. The method of claim 62, wherein saidbiodegradable scaffold is a polymeric biodegradable scaffold.
 64. Themethod of claim 63, wherein said polymeric biodegradable scaffoldcomprising cross-linked dextran, arabinogalactan, chitosan,polyactide-glycolide, alginates, pullulan, gelatine, collagen, fibrin,fibrinogen or albumin.
 65. The method of claim 56, wherein the diameterof said scaffold is 4-10000 um.
 66. The method of claim 56, wherein saidscaffold is degradable upon exposure to predetermined environmentalconditions.
 67. The method of claim 56, wherein said scaffold is anon-biodegradable scaffold.
 68. The method of claim 56, wherein saidscaffold comprises macromolecules necessary for cell growth,morphogenesis, differentiation, or tissue building and combinationsthereof.
 69. The method of claim 68, wherein said macromoleculesnecessary for cell growth is a bone morphogenic protein, a bonemorphogenic-like protein, an epidermal growth factor, a fibroblastgrowth factor, a platelet derived growth factor, an insulin like growthfactor, a transforming growth factor, a vascular endothelial growthfactor, Ang1, PIGF and combinations thereof.
 70. The method of claim 56,wherein said scaffold contains also a therapeutic agent.
 71. The methodof claim 70, wherein said therapeutic agent is an anti-infective, ahormone, an analgesic, an anti-inflammatory agent, a chemotherapeuticagent, an anti-rejection agent, a prostaglandin, RGD peptide andcombinations thereof.
 72. The method of claim 56, wherein said cell isengineered to express a therapeutic agent and/or a macromoleculenecessary for cell growth, morphogenesis, differentiation, or tissuebuilding and combinations thereof.
 73. The method of claim 72, whereinsaid therapeutic agent is anti-infective, a hormone, an analgesic, ananti-inflammatory agent, a chemotherapeutic agent, an anti-rejectionagent, a prostaglandin, RGD peptide and combinations thereof.
 74. Themethod of claim 72, wherein said macromolecule necessary for cellgrowth, morphogenesis, differentiation, or tissue building andcombinations thereof is a bone morphogenic protein, a bonemorphogenic-like protein, an epidermal growth factor, a fibroblastgrowth factor, a platelet derived growth factor, an insulin like growthfactor, a transforming growth factor, a vascular endothelial growthfactor, Ang1, PIGF and combinations thereof.
 75. The method of claim 56,wherein said cell is a mesenchymal stem cell, mesodermal progenitorcell, endothelial precursor cell or neonatal dermal micro vascularendothelial cells (foreskin).
 76. The method of claim 56, wherein saidcell is capable of differentiating into an endotelial cell.
 77. Themethod of claim 56, wherein said cell is isolated from peripheral blood.78. The method of claim 56, wherein said cell is isolated from mammalianartery, vein or capillary.
 79. The method of claim 56, wherein said cellis genetically engineered to express a reporter gene.
 80. The method ofclaim 79, wherein the reporter gene is a fluorescent protein, Luciferaseor b-gal.
 81. The method of claim 56, wherein said step of growingscaffold encircled by cells so as to form a tubularized tissue is by theuse of a bioreactor.
 82. The method of claim 56, wherein the tubularizedtissue is a single vessel, composition of several vessels or a capillarynetwork.
 83. A method of regulating angiogenesis comprisingadministering to said patient host an effective amount of thetubularized tissue of claim 1, so as to promoting angiogenesis.
 84. Amethod of regulating vasculogenesis in a tissue comprising administeringto said patient host an effective amount of the tubularized tissue ofclaim 1, so as to promoting vasculogenesis.
 85. A method of treatinghypoxia, avascular necrosis, foot ulcer or gangrene result fromdiabetes, athersclerosis, stroke, wound, fracture of bone, comprisingadministering to said patient host an effective amount of thetubularized tissue of claim 1 so as to increasing the vascularity of atissue thereby treating.
 86. A method of replacing a damaged bloodvessel of a patient comprising the step of administering to the patientan effective amount of the tubularized tissue, thereby replacing damagedblood vessels.
 87. The method of claim 86, wherein the blood vessel is athick blood vessels or a thin blood vessel.
 88. A method for inducingthe formation of new blood vessels in an ischemic tissue in a patient inneed thereof, comprising: administering to said patient host aneffective amount of the tubularized tissue of claim 1, thereby inducingthe formation of new blood vessels.
 89. The method of claim 88, whereinsaid patient is in need of treatment for cerebrovascular ischemia, renalischemia, pulmonary ischemia, limb ischemia, ischemic cardiomyopathy andmyocardial ischemia.
 90. A method of enabling ex vivo organ engineeringby introducing the tubularized tissue of claim 1 to an engineered tissueso as to enable a constant oxygen supply to said engineered tissue. 91.A method of engrafting in a patient in need thereof, comprisingadministering to said patient host an effective amount of thetubularized tissue of claim 1, thereby engrafting in a patient in needthereof.
 92. A method of determining cellular or tissue function of anagent comprising the following steps: obtaining the tubularized tissueof claim 1; contacting said tubularized tissue with an agent, so as toobtain a tubularized tissue contacted with an agent; comparing cellularor tissue function of said tubularized tissue contacted with an agent,to a tubularized tissue, thereby determining cellular or tissue functionof the agent.
 93. The method of claim 92, wherein the cellular or tissuefunction is proliferation, apoptosis, toxicity and differentiation. 94.A method of screening candidate genes which are involved in cellular ortissue function, said method comprising the steps of: obtaining thetubularized tissue of claim 1; obtaining mRNA from said tubularizedtissue and said cell; synthesizing cDNA from said mRNA; amplifying saidcDNA-hybrid, so as to obtain an amplified product; detecting saidamplified product; and comparing said amplified products from saidtubularized tissue and said cell, thereby identifying candidate nucleicacid sequence which is involved in the cellular or tissue function. 95.An ex-vivo system for determining cellular or tissue function of anagent according to the method of claim
 92. 96. An ex-vivo system forscreening candidate genes, which are involved in cellular or tissueaccording to the method of claim
 94. 97. A hybrid tissue made accordingto the steps of: seeding a cell that will form a first tissue type onthe exterior surface of a branched scaffold so as to obtain a scaffoldencircled by cells with spaces in between; adding into said spaces inbetween, a cell that will form a second tissue type; and incubating soas to obtain a hybrid tissue.
 98. The hybrid tissue of claim 97, wherebyin the step of obtaining a hybrid tissue, the hybrid tissue is amonolayer tubular tissue.
 99. The hybrid tissue of claim 97, whereby inthe step of obtaining a hybrid tissue, the hybrid tissue is a multilayertubular tissue.
 100. The hybrid tissue of claim 97, whereby in the stepof obtaining a hybrid tissue, the hybrid tissue is a multilayer hybridtissue comprising more than one type of cells.
 101. The hybrid tissue ofclaim 97, whereby in the step of obtaining a hybrid tissue, said hybridtissue is a vascularized tissue.
 102. The hybrid tissue of claim 97,whereby in the step of seeding a cell on the exterior surface ofscaffold, said scaffold is in the form of a string.
 103. The hybridtissue of claim 97, whereby in the step of seeding a cell on theexterior surface of scaffold, said scaffold is a biodegradable scaffold.104. The hybrid tissue of claim 103, wherein the biodegradable scaffoldis a polymeric biodegradable scaffold.
 105. The hybrid tissue of claim104, wherein the polymeric biodegradable scaffold comprisingcross-linked dextran, arabinogalactan, chitosan, polyactide-glycolide,alginates, pullulan, gelatine, collagen, fibrin, fibrinogen or albumin.106. The hybrid tissue of claim 97, whereby in the step of obtaining ascaffold, the diameter of said scaffold is 4-500 um.
 107. The hybridtissue of claim 97, whereby in the step of obtaining a scaffold, saidscaffold is degradable upon exposure to predetermined environmentalconditions.
 108. The hybrid tissue of claim 97, whereby in the step ofobtaining a scaffold, said scaffold is a non-biodegradable scaffold.109. The hybrid tissue of claim 97, whereby in the step of obtaining ascaffold, said scaffold comprises macromolecules necessary for cellgrowth, morphogenesis, differentiation, or tissue building andcombinations thereof.
 110. The hybrid tissue of claim 109, wherein themacromolecules necessary for cell growth is a bone morphogenic protein,a bone morphogenic-like protein, an epidermal growth factor, afibroblast growth factor, a platelet derived growth factor, an insulinlike growth factor, a transforming growth factor, a vascular endothelialgrowth factor, Ang1, PIGF and combinations thereof.
 111. The hybridtissue of claim 97, whereby in the step of obtaining a scaffold, saidscaffold contains also a therapeutic agent.
 112. The hybrid tissue ofclaim 111, wherein said therapeutic agent is an anti-infective, ahormone, an analgesic, an anti-inflammatory agent, a chemotherapeuticagent, an anti-rejection agent, a prostaglandin, RGD peptide andcombinations thereof.
 113. The hybrid tissue of claim 111, wherein saidtherapeutic agent is an a nucleic acid sequence which encodes ananti-infective, a hormone, an analgesic, an anti-inflammatory agent, achemotherapeutic agent, an anti-rejection agent, a prostaglandin, RGDpeptide and combinations thereof.
 114. The hybrid tissue of claim 97,wherein said cell is engineered to express a therapeutic agent and/or amacromolecule necessary for cell growth, morphogenesis, differentiation,or tissue building and combinations thereof.
 115. The hybrid tissue ofclaim 114, wherein said therapeutic agent is anti-infective, a hormone,an analgesic, an anti-inflammatory agent, a chemotherapeutic agent, ananti-rejection agent, a prostaglandin, RGD peptide and combinationsthereof.
 116. The hybrid tissue of claim 114, wherein said macromoleculenecessary for cell growth, morphogenesis, differentiation, or tissuebuilding and combinations thereof is a bone morphogenic protein, a bonemorphogenic-like protein, an epidermal growth factor, a fibroblastgrowth factor, a platelet derived growth factor, an insulin like growthfactor, a transforming growth factor, a vascular endothelial growthfactor, Ang1, PIGF and combinations thereof.
 117. The hybrid tissue ofclaim 97, whereby in the step of obtaining a cell, said cell is amesenchymal stem cell, mesodermal progenitor cell, endothelial precursorcell or neonatal dermal micro vascular endothelial cells.
 118. Thehybrid tissue of claim 97, whereby in the step of obtaining a cell, saidcell is capable of differentiating into an endotelial cell.
 119. Thehybrid tissue of claim 97, whereby in the step of obtaining a cell, saidcell is isolated from peripheral blood.
 120. The hybrid tissue of claim97, whereby in the step of obtaining a cell, said cell is isolated frommammalian artery, vein or capillary.
 121. The hybrid tissue of claim 97,whereby in the step of obtaining a cell said cell is geneticallyengineered to express a reporter gene.
 122. The hybrid tissue of claim121, wherein the reporter gene is a fluorescent protein, Luciferase orb-gal.
 123. The hybrid tissue of claim 97, wherein said step of growingscaffold encircled by cells so as to form a hybrid tissue is by the useof a bioreactor.
 124. The hybrid tissue of claim 97, wherein the hybridtissue is a single vessel, composition of several vessels or a capillarynetwork.